1. Field of the Invention
This invention pertains to a novel 1,2-dioxetane substrate for use in a chemiluminescent assay for the detection of neuraminidase. This invention takes advantage of the high sensitivity of chemiluminescent 1,2-dioxetane reagents to overcome sensitivity problems encountered in the prior art. Additionally, this invention pertains to methods and kits employing such dioxetanes.
2. Background of the Prior Art
A wide variety of diseases and infections are caused by viruses. Of all of these known diseases and infections, respiratory infections such as those caused by influenza viruses are the most common. Acute respiratory infections can be fatal, especially in elderly patients. Consequently, the development of assays for the detection of viruses and viral infections has become increasingly important.
In general, influenza viruses express surface glycoproteins that have neuraminidase activity. The enzyme neuraminidase, also known as sialidase, is a well-characterized hydrolytic enzyme that has an optimum pH at 5.5 and hydrolyzes substrates that contain 2-ketosidically linked n-acetylneuraminic acids (Neu5ac, also known as sialic acid). This low optimum pH (5.5) for the neuraminidase enzyme makes it difficult to obtain an assay for neuraminidase that has adequate sensitivity.
The detection of neuraminidase is important because neuraminidase is implicated in a variety of biological events. For example, a deficiency in this enzyme leads to sialidosis, an autosomal recessive trait. Additionally, it is known that the release of neuraminidase mediates the penetration of cells by influenza viruses.
Because the early detection of influenza viruses allows for a more effective treatment, it is desirable to have a highly sensitive assay for the early detection of influenza viruses. However, it is very difficult to detect influenza viruses at an early stage using conventional technology because the clinical samples obtained often do not have a sufficient amount of neuraminidase present to be able to be detected by current technology.
A chromogenic/fluorogenic neuraminidase substrate has been developed and is reported in U.S. Pat. No. 5,719,020, Liav, et al. The same is incorporated herein by reference. While the substrate and assay provided in this reference offers some enhancement of specificity and reliability in detection, in fact, the use of chromogenic or fluorogenic reporter molecules suffers from a variety of drawbacks in detection mechanisms, note, for instance, the detailed collection and assessment steps necessary in the assay described in U.S. Pat. No. 5,719,020. A simpler, more reliable, quantifiable detection system is desirable. The problems with specificity for this specific assay are also discussed in Reinhard et al., Biol. Chem., Volume 373, pages 63-68 (1992).
Chemiluminescent substrates release light for a positive indication of the presence of a particular substance in a sample. Chemiluminescent assays which utilize these chemiluminescent substrates are attractive assays because they avoid the need for special procedures for using and discarding radioactive materials. Additionally, chemiluminescent assays typically do not require complicated or involved apparatus for detection of the assayed substance. Further, chemiluminescent assays may be enhanced by water soluble enhancers to enhance the total luminosity. Typical enhancers are set forth in U.S. Pat. No. 5,145,772, incorporated herein by reference.
The assignee of this application, Tropix, Inc., has developed a wide array of chemiluminescent enzyme substrates for use in detection assays, many of which utilize 1,2-dioxetanes. Representative patents addressing these chemiluminescent enzyme substrates include U.S. Pat. Nos. 4,931,223; 4,931,569; 4,952,707; 4,956,477; 4,978,614; 5,032,381; to 5,112,960; 5,145,772; 5,220,005; 5,225,584; 5,326,882; 5,330,900; 5,336,596; 5,869,699; 5,538,847; and 5,871,938, all of which are incorporated herein by reference.
The above-referenced patents address 1,2-dioxetanes which are stabilized by a polycyclic group bonded to one of the carbons in the four membered ring portion of the dioxetane by a Spiro linkage. An electron-rich moiety, typically an aryl group, a phenyl, or a naphthyl group, is bonded to the remaining carbon of the dioxetane ring. Attached to this moiety is an enzyme-cleavable group. When this group is cleaved, an anion is generated which decomposes, causing the dioxetane to release light. In addition, the carbon that bears the above-identified electron-rich moiety may also bear an alkoxy or other electron-active group.
As disclosed in U.S. Pat. No. 5,112,960, an enzyme-triggerable dioxetane such as 3-(4-methoxy-spiro[1,2-dioxetane-3,2xe2x80x2-tricyclo[3.3.1.13,7]decan]-4-yl-phenyl phosphate and its salts (AMPPD(copyright)) is a highly effective reporter molecule. Superior performance can be obtained by selective substitution on the polycyclic group. For example, substitution with an electron-active species, such as chlorine, has been shown to dramatically improve reaction speed and signal-to-noise ratio (S/N). The chlorine-substituted counterpart of AMPPD(copyright), CSPD(copyright), has been widely commercialized by Tropix, Inc. xe2x80x9cThird-generationxe2x80x9d dioxetane compounds of similar structure, wherein the phenyl or naphthyl moiety also bears an electron-active substituent, such as chlorine, offer further improvements in performance. These xe2x80x9cthird generationxe2x80x9d dioxetanes have also been commercialized by Tropix, Inc. The phosphate moieties are available under the trademarks CDP(copyright) and CDP-STAR(copyright). These reporter molecules, which are chemiluminescent in nature, are referred to as enzyme-triggerable dioxetanes. To date, alkaline phosphatase has been the dominant enzyme of interest as a triggering agent.
Although much is known about chemiluminescent assays generally, the existing literature does not describe a triggerable dioxetane which is specific for the neuraminidase enzyme. Furthermore, the existing literature does not disclose a chemiluminescent detection assay, or a substrate for use in such an assay, for the sensitive detection of neuraminidase. Accordingly, a need exists for a 1,2-dioxetane compound which can be used to detect the presence of neuraminidase. Thus, it remains a goal of one of ordinary skill in the art to find an assay to detect the presence of the neuraminidase enzyme which is highly sensitive and employs reagents which can be obtained through simplified procedures.
The above objects, and other discussed in more detail below, are met by a chemiluminescent assay which relies on chemiluminescent 1,2-dioxetanes. Other dioxetanes, developed by the assignee here, Tropix, Inc., are the subject of a wide variety of United States patents. The 1,2-dioxetane substrates useful in the present invention are generally represented by the following formula: 
wherein T is a substituted or unsubstituted polycycloalkyl group bonded to the 4-membered ring portion of said dioxetane by a spiro linkage, said substituents being independently selected from the group consisting of a hydroxyl group, fluorine, chlorine, an unsubstituted straight or branched chain alkyl group of 1-6 carbon atoms, a 1-6 carbon alkyl group mono-, di- or tri- substituted with a hydroxy or 1-3 halogen atoms, a phenyl group, a cyano group and an amide group;
wherein X is selected from the group consisting of phenyl, naphthyl and other heteroaryls, and wherein X bears 1-3 electron active substituents, each electron active substituent being independently selected from the group consisting of halogen (particularly F and Cl), alkoxy, aryloxy, trialkylammonium, alkylamido, arylamido, arylcarbamoyl, alkylcarbamoyl, cyano, nitro, ester, alkylsulfonamido, arylsulfonamido, triphorylmethyl, aryl, alkyl, trialkyl, triarylsilyl, alkylarylsilyl, alkylamidosulfonyl, arylamidosulfonyl, alkylsulfonyl, arylsulfonyl, alkylthioether and arylthioether, and wherein, each alkyl or aryl moiety comprises 1-12 carbon atoms;
wherein Z is an enzymatically cleavable group of formula II: 
and R1-R3 are hydrogen or alkyl groups (straight chain or branched) of 1-4 carbon atoms;
and wherein R is an alkyl, aryl, aralkyl or cycloalkyl of 1-20 carbon atoms, which may contain 1-2 hetero atoms selected from the group consisting of phosphorus, nitrogen, sulfur and oxygen; and wherein R can bear at least one halogen substituent.
It is another object of this invention to provide diagnostic kits and methods for detecting neuraminidase employing such substrates.